Project Abstract The main aim of this proposal is to fully evaluate the potential role of 3'-untranslated region of the adeno- associated virus serotype 2 (AAV2) genome in the oncogenic insertional mutagenesis and eventually in inducing hepatocellular carcinoma (HCC). AAV2 is considered a non-pathogenic virus for the past half century because more than 90% of the human population is AAV2 seropositive, and yet no known-disease has been associated with the viral infection. In addition, since the late 1990s, recombinant AAV vectors have been extensively studied and developed as a system of gene delivery to treat a number of genetic diseases both in the laboratory and in the clinic, and thus far, no adverse event has ever been reported. However, the above well-known knowledge has recently been questioned by a clinical observation. In 11 of 193 human HCC samples, clonal integrations of partial wild-type (WT) AAV2 genome containing only the 3'-untranslated region were observed. We have had a long-term interest in AAV biology in general, and in the 3'-untranslated region of AAV genome in particular. More than two decades ago, we elucidated the role of the D-sequence in the life cycle of the WT AAV2, including genome rescue, replication and encapsidation. Its influence on rAAV transgene expression was also examined, which eventually led to the development of D-sequence-substituted single-stranded rAAV vectors as well as D-sequence-deleted single-stranded DNA mini-vectors. Earlier this year, we developed polyA-deleted WT AAV and rAAV vectors and provided evidence for a productive life cycle of WT AAV2 in the complete absence of a conventional polyA signal. More recently, we substituted the entire ITR2 with ITRs from other AAV serotypes to produce high-titer, high-potency rAAV vectors. Although we have extensively studied the role of polyA signal, D-sequence and various ITRs, their functions in the viral genome integration, or in the induction of HCC has not been rigorously examined. In addition, the patterns of AAV integration has not been revealed by high-throughput methods in human liver cells in vitro, or in primary human hepatocytes xenografted mice in vivo, a valuable, clinically relevant liver model. Both our and others publications indicated that this model has significant potential for gene therapy applications. We also have designed additional strategies to overcome the variables of this new animal model that may influence experimental outcomes. Thus, in this proposal, we will pursue the following Specific Aims and related hypotheses: Specific Aim 1: Role of the 3'-untranslated region in AAV viral genome integration in vitro. Specific Aim 2: Role of the 3'-untranslated region in AAV-mediated of human HCC initiation in vivo. These studies will not only establish a template animal model for studying viral oncogenic insertional mutagenesis, but also the knowledge gained from these studies will be applicable in the design of safer AAV vectors for gene therapy studies in the future.